Am J Cardiovasc Drugs 2011; 11 (2): 73-82

CURRENT OPINION 1175-3277/11/0002-0073/$49.95/0

ª 2011 Adis Data Information BV. All rights reserved.

Ozone: A New Therapeutic Agent in Vascular Diseases

Velio Bocci,1 Iacopo Zanardi2 and Valter Travagli2
1 Department of Physiology, University of Siena, Siena, Italy
2 Department of Pharmaceutical Chemistry and Technology, University of Siena, Siena, Italy

Abstract In this article, we scientifically evaluate the bio-oxidative procedure known as oxygen-ozone therapy.
Research over a decade has established a comprehensive framework for understanding and recommending
this type of autohemotherapy in vascular diseases. In contrast, a non-specific immunomodulation therapy,
using heavily oxidized and denatured blood, has been recently used in studies involving a total of approx-
imately 3000 patients and has led to ‘disappointing’ results. Such a treatment appears to be an inappropriate
example of the so-called minor autohemotherapy, and its poor outcomes may discourage any further
studies. Therefore it appears necessary to clarify that the use of only a minimal ozone dose and a valid
experimental protocol is likely to produce beneficial results. Millions of people suffer from chronic limb,
brain, and heart ischemia, and such patients may benefit if appropriate ozone therapy could be implemented.
Accordingly, we propose the need for a well designed, multicenter, clinical trial to be conducted.

1. Introduction modulation therapy (IMT) using heavily oxidized blood has

yielded ‘disappointing’ results in the treatment of functional
Statins, platelet aggregation inhibitors, antidiabetic agents, class II–IV chronic heart failure (CHF).[9]
anticoagulants, antihypertensives, vasodilators, and antiar- The present review has four objectives: firstly, to inform
rhythmic agents are drugs commonly used every day by millions clinical scientists and physicians that using ozone therapy is no
of patients, while complementary medications recommended longer an empirical approach, but rather it is based on a
by phytotherapists and homeopaths are far less popular be- framework built on a sequence of biochemical reactions leading
cause their mechanisms of action, safety, and efficacy remain to well defined biological effects on both blood cells and the
uncertain. While oxygen is well known as a useful medical organism. Secondly, careful scrutiny[10] has excluded any po-
gas,[1] the combination with ozone, namely oxygen-ozone tentially toxic effects, by using small ozone dosages within a
therapy or simply ozone therapy, has been used less often and precisely determined therapeutic range. Thirdly, by using
with mixed results. Bio-oxidative approaches to the treatment standardized procedures, the usefulness of ozone therapy in
of diseases have been known from the beginning of the 20th PAD has already been shown by several authors.[2-8] Fourthly,
century, but UV blood irradiation used in infectious diseases in in open contrast, the misuse of an excessive amount of ozone
the pre-antibiotic era has fallen out of use because it is difficult combined with UV irradiation and heat stress on blood has
to standardize and cumbersome to perform. totally compromised its efficacy in improving the prognosis of
Oxygen-ozone therapy has come of age in the last three de- CHF.[9] Therefore this article may inform readers that ozone
cades, with ozone representing the therapeutic agent, while therapy is a rational treatment for vascular diseases provided
oxygen is simply used as the feeding gas for ozone generation. that ozone is used as a ‘real drug’ and not as a toxic gas.
Ozone is one of the most reactive gases and has a spontaneous
half-life of 40 minutes at 20C. In spite of the extemporary use,
modern medical ozone generators produce precisely determined 2. The Mechanisms of Action of Ozone in
ozone concentrations in real time. The medical literature on Contact with Blood
ozone therapy is modest, although it has been shown since 1981
to be more useful than pentoxyfilline and prostanoid analogs in Ozone is known chemically as the third most reactive gas,
peripheral arterial disease (PAD).[2-8] In contrast, immuno- with an oxidation potential (E) of +2.08 V, while hydrogen
74 Bocci et al.

peroxide (H2O2), produced by several oxido-reductases known Ozone

as nicotinamide adenine dinucleotide phosphate oxidases

(NOXs), has an E of +1.78 V. Moreover, H2O2 is physiologi- Aqueous environment of plasma

cally generated in the mitochondria following dismutation of

superoxide anion (O2 -) produced by the mitochondrial com-
plex III. By comparison, hypochlorite has an E of +1.48 V. ROS
(early phase)
LOPs
(late phase)
On this basis, it is not surprising that, until recently, chemists
have considered ozone a toxic gas that should not be used in Blood cells Albumin adducts
medicine.[11,12] Indeed, there is ample literature and knowledge
showing the toxicity of ozone to the pulmonary system.[13-16] Fig. 1. A schematic view of ozone reactivity in contact with blood.
LOPs = lipid oxidation products; ROS = reactive oxygen species.
Although this is correct, it has also led to an incorrect con-
clusion. The surface of the human lungs (about 70 m2) is cov- such as nitric oxide (NO), carbon monoxide (CO), and hydro-
ered with a very thin liquid film (0.1 mm)[17] consisting of a small gen sulfide (H2S), produced under physiological conditions and
volume (about 20 mL) of epithelial lining fluid (ELF), which acting as signaling molecules, are considered essential for life
contains a small amount of antioxidants. It is obvious that and are categorized as medical gases, together with hydrogen
individuals inhaling ozone-contaminated air over a period of (H2) and xenon (Xe).[1,25] The second one is that micromolar,
months experience a continuous and cumulative toxicity due to or even submicromolar, amounts of H2O2 and LOPs such as
exposure to many grams of ozone, capable of inducing an 4-hydroxynonenal (4-HNE), from long-chain n-6 polyunsaturated
enormous production of reactive oxygen species (ROS). These fatty acids (PUFAs), and a smaller amount of trans-4-hydroxy-
compounds cause local and generalized damage, by generating 2-hexenal (4-HHE)[26] are valued as crucial signaling molecules
a local inflammation amplified by the release of lipid oxidation capable of switching on cell proliferation and differentiation,
products (LOPs), and pro-inflammatory cytokines (tumor ne- the production of some cytokines, and the upregulation of a
crosis factor [TNF]-a, interleukin [IL]-1, IL-6, etc.) released by number of antioxidant enzymes such as superoxide dismutase
an excess of macrophages and neutrophils.[18,19] It must also be (SOD), catalase, glutathione (GSH)-peroxidases, GSH-reduc-
clarified that ozone acts only and always on the surface of the tase, and GSH-transferases, and of a very protective enzyme
ELF and never enters the epithelial cells of any tissue, because it such as heme-oxygenase-1 (HO-1). It follows that the concept
reacts in a matter of seconds and only within the superficial of ozone as a strong oxidant as well as a therapeutic agent has
aqueous film.[20] The damaging effect of ozone, linked to the been accepted by the scientific community.[27]
generated toxic compounds, has led to the belief that ozone is The following reaction:
always toxic, but this is not true when an appropriate ozone
3O2 þ 68; 400 Cal#2O3
therapy procedure is followed: in this case, a small volume of
gas mixture composed of oxygen (‡95%)-ozone (£5%) comes shows that ozone in the aqueous environment of blood, where it
into contact, for only 5 minutes, with an equivalent volume of dissolves very quickly,[28] releases its energy to hematic com-
blood, which in comparison to the ELF has a far higher anti- ponents, and hence into the body during reinfusion into the
oxidant capacity able to easily quench ozone reactivity. An blood donor. Ozone never enters into the circulation because it
extensive review of this topic has been previously published.[21] is totally consumed either ex vivo in the glass bottle, or after
Among the numerous approaches classified within comple- triggering the reaction on the surface of the skin and mucosae.
mentary medicine, there are several with obscure mechanisms Most of the basic chemical reactions involved have been clari-
of action, perhaps because our current analytical method- fied over the last 15 years.[27,29-34] The therapeutic range has
ologies are unable to decipher them. On the other hand, ozone been precisely defined to be within ozone concentrations of
therapy has been recorded among the bio-oxidative therapies 20–80 mg/mL (0.42–1.68 mmol/mL of gas [pure O2: 95% and
by the National Center for Complementary and Alternative O3: 5%]) per mL of human blood. An ozone concentration
Medicine of the National Institutes of Health.[22] It has been below 10 mg/mL of gas per mL of blood (0.21 mmol/mL) is in most
possible to elucidate and to show that, once ozone has dissolved cases totally quenched by the hydrosoluble antioxidants and
in the aqueous environment of plasma, it gives rise to a series of therefore no biological effects can be elicited and, at best, the
reactions, as shown in figure 1, that are well understood bio- procedure has a placebo effect only. Owing to the potent anti-
chemically. Moreover, currently two important paradoxes have oxidant capacity of blood, due to its hydrophilic and lipophilic
been reviewed:[23,24] the first one is that small amounts of gases antioxidants and cellular enzymes, a small part of the ozone

ª 2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (2)
Ozone Therapy in Vascular Diseases 75

dose dissolved in the aqueous environment is instantly Most importantly, H2O2, which has no electric charge,
quenched by free antioxidants (mainly uric acid, ascorbic acid, rapidly enters the blood cells, and the chemical gradient be-
GSH, cysteine, and albumin), while the bulk of the ozone reacts tween plasma and cells has been measured, with intracellular
with PUFAs mostly present in the three hydrophobic binding levels being about 10% of the extracellular concentration.[43,44]
domains of albumin.[10] Lipoproteins are not targeted by ozone. In other words, when the highest ozone concentration is mixed
The fundamental reaction follows: with blood, depending upon the modest interindividual vari-
-R-CH¼CH-R þ H2 O þ O3 #2 RCHO þ H2 O2 ability of antioxidant potency (1.28–1.83 mmol/L plasma),[45]
the highest H2O2 concentration measured in plasma is ~40 mM,[46]
Thus the potential energy of ozone is finally transferred into two
and therefore inside the cells is at most 4 mM. The sudden influx
fundamental messengers such as H2O2, as a ROS, and aldehydic
of this small amount of H2O2 into blood cells provides the
molecules, of which 4-HNE and 4-HHE are the relevant LOPs:
essential stimulus to activate a series of biochemical reactions.
Water of plasma þ O3 #H2 O2 þ 4-HNE þ HHE
Given the high reactivity of ozone, these biochemical re- 2.2 Biochemical Reactions in Erythrocytes
actions occur in a few seconds, and, in fact, within 5 minutes
of mixing an average of 200 mL of human blood ex vivo in a As a first consequence, ozone therapy leads to the activation
sterile glass bottle with 200 mL of gas mixture (O2 + O3), the of glycolysis with an increase in adenosine triphosphate (ATP)
ozone is totally exhausted while the ~95% oxygen, dissolved in and 2,3-diphosphoglycerate (2,3-DPG).[27] Functionally, the
the plasma water, fully saturates hemoglobin: sigmoidal oxygen-binding curve of hemoglobin shifts to the right
Hb4 O4-6 þ O2 #Hb4 O8 and increases the release of oxygen at the tissue level. The
erythrocytes mop up most of the H2O2 and promptly reduce it to
Blood pO2 up to ~400 mmHg in the bottle is useful, but it is
water through the action of GSH. The sudden formation of
only of small practical relevance because oxygenated-ozonated
GSSG alters the GSH/GSSG ratio within the cell. However, this
blood is normally reinfused via the venous route during the
is rapidly corrected by either expelling some of the GSSG or by
subsequent 20 minutes and is heavily diluted with venous
reducing it via GSH reductase at the expense of either ascorbic
blood. Therefore, ozone represents the therapeutic agent while
acid or thioredoxin, which has two free sulfhydryl groups. This
the oxygen is only necessary for generating the ozone.
important recycling process occurs within about 3 minutes.[40]
Alternatively, activation of glucose-6-phosphate dehydrogenase
2.1 What is the Fate of H2O2? (G6PDH) provides reducing power and activates glycolysis.[34]

Our work[10,35,36] and that of Shinriki et al.[37] has verified

that depending upon the ozone dosage, H2O2 in plasma oxi- 2.3 Biochemical Reactions in Leukocytes
dizes most of the ascorbic acid to dehydroascorbate and about
Following ozone therapy, neutrophil phagocytic activity is
half of the uric acid to allantoin, while bilirubin remains un-
enhanced.[27] Inside monocytes and lymphocytes, H2O2 acti-
altered. Whether a trace of GSH is oxidized to a sulfonic acid
vates a tyrosine kinase with consequent phosphorylation of
remains undetermined and, similarly, so far we have not iden-
IkB, one of the components of the nuclear factor-kB (NF-kB)
tified whether the sulfhydryl (SH) group of albumin is oxidized
trimeric complex.[47] The phosphorylated IkB detaches from
to sulfenic acid:[38,39]
the complex and is degraded in the proteasome. The remaining
R-SH þ H2 O2 #RSOH heterodimer p50-p65 is transferred into the nucleus where it can
During the initial rapid multiple reactions of ozone with plas- activate up to ~100 genes. Of particular significance is the final
ma components, a variable amount of the ozone is obliterated by release of a number of cytokines (interferon [IFN]-g and IL-8)
the abundance of hydrophilic antioxidants. However, it is note- and acute-phase proteins.[27]
worthy that, with the exception of uric acid, dehydroascorbate
and oxidized glutathione (GSSG) are reduced back to their nor- 2.4 Biochemical Reactions in Platelets
mal levels in less than 20 minutes. This is achieved through the
efficiency of the recycling system based on a multitude of reducing In response to the ozone concentration, the release of platelet-
molecules including a-lipoate, vitamin E, thioredoxin, and nic- derived growth factor (PDGF)-AB, transforming growth factor
otinamide adenine dinucleotide phosphate[40,41] (NADPH), and (TGF) b-1, and IL-8 have been measured.[48] Growth factors
involving a well coordinated sequence of electron donations.[42] have specific relevance in the enhancement of ulcer healing in PAD.

ª 2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (2)
76 Bocci et al.

It must be said that the H2O2 concentration in the cells of 4-HNE is an enlightening example of how the normal serum
(4 mM) is the minimum necessary to switch on cellular responses, level of a potentially toxic aldehyde produced by physiological
and it probably lasts for a few seconds as GSH peroxidases, peroxidation can activate a number of useful signaling path-
peroxiredoxin, and catalase promptly reduce it to H2O. In ways.[58]
plasma, the H2O2 half-life is less than 1 minute, and it is absent At the time of ozonated blood infusion, 4-HNE-Cys adduct
during blood reinfusion.[32] can also act on the vast expanse of endothelial cells and, by
stimulating endothelial nitric oxide synthase (eNOS), enhance
2.5 What is the Fate of Lipid Oxidation Products? the biosynthesis of NO via the 5-electron oxidation of L-argi-
nine. This crucial mediator acts on the enzyme-soluble guanyl-
Among a variety of LOPs, newly formed lipoperoxide rad- ate cyclase able to catalyze the conversion of guanosine
icals are rapidly reduced to hydroperoxide, while among a va- triphosphate (GTP) to cyclic guanosine monophosphate
riety of alkenals, the bulk is represented by the fairly stable (cGMP), which is the second messenger involved in crucial
4-HNE.[35] This compound is an amphipathic molecule react- physiological processes including the regulation of vascular
ing with free GSH, carnosine, and mainly albumin. Although smooth muscle tone and inhibition of platelet aggregation,
4-HNE is a toxic aldehyde generated by ozone, it eventually smooth muscle proliferation, and monocyte adhesion to the
acts as a useful messenger through three processes schemati- vascular endothelial cells.[59,60] Thus, the enhanced endogenous
cally indicated as: (1) detoxification, (2) dilution, and (3) ex- formation of NO and S-nitrosothiols is one of the bases of
cretion. Specifically: ozone therapy for preventing and treating hypertension, stroke,
1. A small amount of 4-HNE is broken down immediately by and coronary infarction. Indeed, in our clinical trial in PAD it
enzymes such as GSH-S-transferases and aldehyde dehydrogenase was shown to be better than the orthodox infusion of iloprost.[8]
or by other detoxifying enzymes as described by Awasthi NO, S-nitrosothiols, and a trace of CO released with bilirubin
et al.[49] via the upregulation of HO-1 activity allows vasodilation, thus
2. The bulk is bound to the -SH group of Cys34 present in improving tissue oxygenation in ischemic tissues. Moreover, an
domain-I of albumin but also to free GSH. Furthermore, 11 increased production of NO counteracts the excessive endo-
nucleophilic residues present in albumin (including Lys199 and thelial release of O2 - caused by the chronic inflammation
His146) can also bind 11 molecules of 4-HNE.[39] Thus, because typical of atherosclerosis.
of the large amount of albumin (~120 g intravascular and H2S is endogenously synthesized from L-cysteine by cysta-
160–200 g extravascular), the bound alkenals undergo major thionine-b-synthase (CBS) and cystathionine-g-lyase (CSE).
dilution in the body fluids implying a most important loss of Interestingly this is another gas, a potentially toxic molecule,
toxicity. 4-HNE has no membrane receptor but the albumin that when released in trace amounts becomes a further im-
adduct can facilitate its transport throughout the body. portant physiological mediator like NO and CO.[1] At physio-
3. 4-HNE is also excreted into bile and urine after hepatic logical concentrations, H2S seems to modulate the K+-ATP
detoxification[50] and renal excretion as mercapturic acid channel opening involving vascular flow regulation.[1,61]
conjugates.[51] An interesting aspect is that albumin can As far as hydrogen treatment is concerned, it significantly
transport 4-HNE in all body tissues, from liver to endocrine increases antioxidant enzyme activity, decreases lipid peroxide
glands and the CNS. Thus 4-HNE-Cys adducts can be released levels, and decreases circulating proinflammatory cytokine
at many sites and inform a variety of cells of a transient, acute levels.[1] The therapeutic potential of H2 in correcting ischemia-
oxidative stress. At submicromolar or picomolar levels, 4-HNE reperfusion damage after either coronary infarction or stroke is
can act as a signaling molecule,[52] able to activate the synthesis potentially enormous.[62,63]
of g-glutamate cysteine ligase, g-glutamyl transferase, g-glutamyl Similarly, the minimal amount of ozone necessary to trigger
transpeptidase, HSP-70, HO-1, and antioxidant enzymes such useful biological effects fits very well with the concept of the
as SOD, GSH-peroxidase, catalase, and G6PDH, a critical xenohormesis theory.[64] Another very interesting aspect ob-
enzyme electron-donor during erythropoiesis in the bone served in 67- to 78-year-old subjects affected by the dry form of
marrow. There is a wide consensus on the relevance of the age-related macular degeneration is that a majority of them
induction of protective molecules during small but recurrent report a feeling of euphoria and a sense of wellness and physical
oxidative stress.[53-57] In other words, the concept that a energy throughout the ozone therapy cycle of 14–16 treatments
precisely controlled oxidative stress can strengthen the anti- lasting about 2 months.[30] Whether these feelings are simply
oxidant defenses is well accepted today. The biological behavior due to faith in the medical treatment, i.e. the power of the

ª 2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (2)
Ozone Therapy in Vascular Diseases 77

placebo effect,[65] or are caused by the generated ozone mes- 3. The Reliable Route of Ozone Administration
sengers able to modify or improve the hormonal secretion, is
not yet known. Lack of funding has always prevented per- Ozone can be administered with great flexibility but it is of
forming a study in normal volunteers where, before and after paramount importance to bear in mind that it should never be
ozone therapy, the complete hormonal pattern and cycling in injected intravenously as a gas because of the risk of provoking
the plasma throughout the 24 hours can be determined. This oxygen embolism, given the fact that the gas mixture always
study would be very informative and helpful for understanding contains no less than 95% oxygen. So far, the optimal approach
why the patients experience a feeling of well-being throughout has been major autohemotherapy (AHT) because, on the basis
the ozone therapy course. In our opinion, this may be due to of the patient’s bodyweight, a predetermined volume of blood
improved oxygenation and/or enhanced secretion of growth (from 100 mL up to 225 mL) to which has been added either
hormone (GH), cortisol, and dehydroepiandrosterone (DHEA). sodium citrate 3.8% (1 + 9 mL blood) or heparin (20 IU/mL of
Patients with arthrosis pain have also reported marked im- blood) can be exposed to an equal volume of gas (O2– O3), with
provement and less pain that may be due to release of ACTH- the ozone concentration precisely determined by using an
cortisol or to a limited stimulation of COX-2 with enhanced ozone-resistant, disposable 500 mL glass bottle under vacuum.
release of prostacyclin (PGI2).[66] It is also possible that LOPs It is a simple, inexpensive (all the necessary disposable materials
reaching the hypothalamic area improve the release of neuro- cost about $US12) procedure and it has already yielded ther-
transmitters such as serotonin, dopamine, and endorphins as apeutic results in vascular diseases that are superior to those
has been observed after intense physical exercise.[67] achieved by conventional medicine.[2-8] Moreover, the ther-
By using the above-reported therapeutic range of ozone apeutic modalities have been extended: they include the quasi-
concentrations strictly related to the blood volume, it must be total body exposure to O2– O3[69] and the extracorporeal blood
noted that neither acute nor chronic toxicity has ever been circulation against O2– O3 (EBOO).[8] The latter procedure, to
observed during or after ozone therapy. Such a result seems be used in emergency conditions, is rather invasive because
unbelievable, but further studies[10,33] performed to evaluate blood collected from a vein circulates through an ozone-
possible hematochemical or biochemical and enzymatic mod- resistant gas exchanger[70] and, with the help of a peristaltic
ifications have clearly demonstrated that ozone concentrations pump, returns to the circulation via a contralateral vein. On the
of 20–80 (even up 160) mg/mL (of gas per mL of whole blood) do other hand, the partial cutaneous exposure to oxygen-ozone
not damage blood cells or other components. Indeed, during does not need any venous puncture and, owing to the vast ex-
the ozonation process no more than 35% of the total anti- panse of the skin, allows a generalized and beneficial effect.
oxidant capacity is oxidized and as mentioned earlier this is Clearly, today we can select the most suitable method for dif-
reconstituted within 20 minutes in vitro and even more rapidly ferent pathologies, their stage, and the patient’s condition.
in vivo. The evaluation of hemolysis after blood ozonation is A separate discussion is needed for minor AHT, which basically
one of the most reliable markers for assessing damage to er- consists of withdrawing 5 mL of blood to be immediately and
ythrocytes: it always remains below 1%[33] and actually a small vigorously mixed for 1 minute with an equal volume of O2/O3 at
quantity of free heme is useful for inducing the upregulation of an ozone concentration ranging between 80 and 100 mg/mL of
HO-1. gas per mL of blood. The slightly oxidized blood, including the
Is it possible that ozone can oxidize the phospholipids and foam, is promptly injected into the gluteus muscle without the
cholesterol of the cell membrane? The demonstration that the need for any anesthetic. As a non-specific immunomodulatory
negative electric charge of the erythrocyte membrane is un- approach, it has been widely used during the last two decades
modified even after ozonation at 160 mg/mL (of ozone per mL for successfully treating herpetic infections.[30] A slight degree
of blood) excludes this possibility.[33] It is well known that of hemolysis (1–2%) is purposefully required because the heme
blood cell membranes are always shielded by a cloud of albu- released in the gluteal muscle will stimulate the synthesis of
min molecules able to prevent any ozone attack, as it may occur HO-1.[71]
as a result of excessive oxidation or may well occur during
ozonation of saline-washed erythrocytes. Unfortunately too 4. Which Diseases are Suitable for Treatment with
many artificial experiments performed with saline-washed cells Ozone Therapy?
totally deprived of the plasmatic antioxidants have led to in-
correct conclusions about a direct effect of ozone on the cell On the basis of the mechanisms of action, ozone therapy can
membrane phospholipids.[68] induce the following biological responses:

ª 2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (2)
78 Bocci et al.

Table I. Results in patients with peripheral arterial disease after ozone therapy[2]
Outcome Fontaine-Leriche stage

II (n = 62) III (n = 51) IV (n = 39)

Very good improvement (% patients) 87.1 70.6 53.8
walking distance (m) >1000 >800 >500
No pain at rest Gangrene healed
Improved (% patients) 9.7 21.6 25.6
walking distance (m) >400–500 >300–400 Amputation of toes with healing of stump
Occasional pain
No improvement or progression (% patients) 3.2 7.8 20.6

 Improvement in blood circulation and oxygen delivery to mation of an ulcer, which will not heal because oxygen, nu-
ischemic tissue as a result of the concerted effect of NO and trients, and growth factors indispensable for the repair process
CO and an increase in intraerythrocytic 2,3-DPG level; are lacking. This pathology is the most suitable for treatment
 enhancement in general metabolism through improved with major AHT. According to the Fontaine-Leriche classi-
oxygen delivery; fication,[73] a patient at either stage II (intermittent claudication
 upregulation of cellular antioxidant enzymes and induction and transitory pain) or stage III (continuous pain, cyanosis,
of HO-1 and HSP-70; and possibly initial ulcers) will achieve the best results. Stage IV
 induction of mild activation of the immune system and includes incipient necrosis of the toes and unbearable pain,
enhanced release of growth factors; leading to surgical amputation that can be avoided with AHT
 excellent disinfectant activity when used topically, but this is in about 50% of cases.[2,3] Table I summarizes a study per-
negligible in the circulation because of the blood antioxidant formed between 1974 and 1980 in 152 patients and shows the
capacity; this property is of value in all cutaneous and beneficial effects of major AHT.[2] In comparison to pentox-
mucosal infections and ulcers, especially in the diabetic foot; yfilline and prostanoids (the standard of orthodox treatment),
 absence of acute or late adverse effects;[72] major AHT has proved to be more effective and without ad-
 a surprising feeling of well-being, probably through stim- verse effects in patients with ischemic vascular disease. Several
ulation of the neuroendocrine system. other small studies[3-7,74-76] have amply confirmed these results.
On this basis it appears that ozone, by acting on many tar- In a small trial using EBOO, 28 patients were randomized to
gets, can indirectly help in recovering functional activities af- receive either their own ozonated blood or an intravenous in-
fected by chronic disease and, if this interpretation is correct, fusion of prostacyclin.[8] All patients continued conventional
ozone therapy acts as a biological response modifier. Although treatment with statins, antihypertensive agents, and platelet
ozone therapy is now used in many countries, it is mostly used aggregation inhibitors. Ozone therapy proved more effective
by private physicians, and the performance of large clinical than prostacyclin in terms of pain reduction and improvement
trials has been severely hampered by a lack of sponsors and in quality of life, but no significant difference was seen in vas-
disinterest of pharmaceutical companies. All these reasons may cularization of the lower limbs between the two groups, most
explain why ozone therapy remains little known to orthodox likely due to the short duration of treatment (14 treatments in
medicine and why it has not been evaluated in cardiovascular 7 weeks). More prolonged treatments have led to a satisfactory
diseases, which represent the ideal pathology. However, a healing of ulcers.[4] It is a mistake to stop therapy too early in
number of studies have been performed with results as de- these patients because major AHT, as with other conventional
scribed in the following sections. drug therapies, must be continued, albeit less frequently, for life.
Topical therapy performed with ozonated olive oil is extremely
4.1 Peripheral Obstructive Arterial Diseases useful when initial dry gangrene or ulcers are present. The fre-
quency of major AHT depends upon the stage of the disease,
Even a modest obstruction of limb arteries due to athero- and with stages III and IV it can be carried out every day in an
sclerosis, diabetes, or Buerger’s disease (thromboangiitis ob- attempt to prevent amputation. How well major AHT works
literans) leads to a progressive reduction of blood flow to the appears evident from the fact that the excruciating nocturnal
feet. Tissue ischemia and any minor trauma facilitate the for- pain disappears after the first two to three treatments,[2,30]

ª 2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (2)
Ozone Therapy in Vascular Diseases 79

indicating an improvement in blood flow in the ischemic tissue of Immune Modulation Therapy in Patients with Symptomatic
and a lack of ‘stealing’ blood away from underperfused muscle. Peripheral Arterial Disease).[81] It is important to note that this
trial had to be stopped 3 months early because it did not show
5. Studies that may have Compromised any improvement in PAD and because of a significantly higher
the Future of Ozone Therapy rate of malignancies in the IMT group. Although in a pilot
study[82] of 73 patients with heart failure, the IMT seemed to
On the basis of very encouraging clinical results achieved result in a reduction in mortality, a subsequent multicenter study
with correctly administered ozone therapy, the possibility that in 2426 patients,[9] called the ACCLAIM (Advanced Chronic
ozone therapy may help patients with CHF must be considered Heart Failure Clinical Assessment Of Immune Modulation
because this is a critical and costly pathology. In the past, we have Therapy) trial, in chronic heart failure produced ‘disappointing’
treated three CHF patients with EBOO. After some 20 treatments, results. In this trial no particular cancer predominated ‘‘although
they showed a marked and consistent improvement, suggesting an imbalance was seen in reports of colorectal cancer’’ (nine
the possible induction of neo-angiogenesis after the mobilization patients in the IMT group and three in the placebo group).
of staminal cells.[7] However, once again we attempted to under- The proponents of IMT[77] support the concept that after
take a randomized trial but were unable to cover the cost of the intramuscular administration of heavily denatured blood, a
gas-exchange device and insurance, and the study was stopped. non-specific immunomodulation ensues with an up-regulation
In the meantime, other clinical trials using a device by in the production of anti-inflammatory cytokines such as IL-10
Vasogen Inc. (Mississauga, ON, Canada) have progressed be- and TGF-b and inhibition of proinflammatory cytokines, such
cause this company was able to support these studies in multiple as TNF-a, IL-1, and IL-6.[9] As CHF and chronic limb ischemia
countries. The outcome of this enterprise may have compro- are conditions linked to inflammation and chronic oxidative
mised the progress of ozone therapy, although clinical scientists stress, in theory the reduction of proinflammatory cytokines
seem not to have noticed the terribly harsh oxidation of blood may down-regulate chronic inflammation and delay the prog-
injected into patients with CHF. ress of the disease. However, other studies performed using anti-
In 1997, Bulmer et al.[77] proposed a new procedure that TNF-a antibodies have failed to improve outcomes in CHF.[9]
involved 10 mL of the patient’s anticoagulated venous blood The earlier failure of the SIMPADICO trial not only was an
(+ 2 mL sodium citrate) being exposed to an oxygen/ozone gas excellent indication of a poor result in CHF, but also indicated
mixture (ozone concentration 15.35 g/m3) delivered into the that a strong immune suppression may favor neoplastic growth.
blood at a flow rate of 240 mL/min and UV light (253.7 nm) at a Sliwa and Ansari[83] pointed out several potential problems but
temperature of 42.5C for about 20 minutes. The treated blood did not comment on the incorrect oxidative technology used.
sample was removed from the system and immediately ad- On the other hand, specific criticisms regarding the irrational
ministered by intragluteal injection back into the patient. technology and deep dissensions with Vasogen’s hypothesis
Later, the procedure used an expensive device (VC7000A have been published.[84-86]
system, Celacade, Vasogen Inc.) that was able to deliver an After two decades of studying the mechanisms of action in
enormously toxic dose of ozone (107.5 mg per mL of blood) blood,[27] the therapeutic range of ozone as a therapeutic agent
plus a further blood oxidation induced by UV irradiation at (0.42/1.68 mmol/mL of ozone per mL of anticoagulated blood)
42.5C. The final ozone dose is about 15 000-fold higher than has been well defined. Ozone is a particularly reactive gas but, if
the average ozone dose used during classic major AHT[78] and judiciously used, it is very useful in vasculopathies because it
the extremely high oxidation of blood causes a complete de- enhances vasodilation, it increases the delivery of both oxygen
naturation of blood components.[10] Two treatments were given and growth factors in ischemic tissues, and it up-regulates sev-
on consecutive days, followed by a third on day 14. Subsequent eral antioxidant enzymes (above all HO-I).[87] Immunomodulation
treatments were given at 4-week (28 days) intervals for at least may be only a small additional factor. The misinterpretation of
22 weeks, for a total of eight injections. The above-described the real mechanisms of action and the obstinate use of an in-
procedure was invented with the aim of establishing a non- correct approach can explain the ‘disappointing’ results and the
specific IMT in the hope of reducing the inflammatory process previous failure in treating chronic limb ischemia in the
and chronic oxidative stress present in vascular diseases. It SIMPADICO trial. Moreover, several case studies have dem-
proved to be ineffective in AIDS trials[79,80] and in a multicen- onstrated the validity and safety of correctly administered
ter, randomized, double-blind, placebo-controlled study in 533 ozone therapy in severe chronic limb ischemia.[2-8,74-76] At
patients with symptomatic PAD, called SIMPADICO (Study present, the situation is worse than before but, surprisingly

ª 2011 Adis Data Information BV. All rights reserved. Am J Cardiovasc Drugs 2011; 11 (2)
80 Bocci et al.

enough, practically no attention has been paid to the fact that Table II. Schedule of ozone treatment in proposed clinical trial for chronic
this clamorous failure was due to an excessive use of ozone. The heart failure
Vasogen procedure was very irrational, not only because it used No. of Ozone Total blood Total ozone
an enormous and toxic dose of ozone, but also because it added treatments concentrations volume as well as dose (mg)
an ambiguous UV irradiation of blood further heat-stressed at (mg per mL gas volume (mL)
of gas mixture)
42.5C for 20 minutes, resulting in the blood being so ‘cooked’
that had to be immediately injected. 1st and 2nd 20a 200 4.0
(week 1)
3rd and 4th 30 200 6.0
6. A Schematic Proposal for a Clinical Trial (week 2)
in Patients with Chronic Heart Failure 5th and 6th 40 200 8.0
(week 3)
This section describes the proposed design for a trial of
7th and 8th 50 200 10.0
ozone therapy in patients with CHF. Patients eligible to enter
(week 4)
the trial would be those classified within the New York Heart
9th to 32nd 60 200 12.0
Association (NYHA) Class II and III heart failure with a left-
a Such a low concentration is useful to avoid an initial excessive
ventricular ejection fraction (LVEF) of 30% or less, and who oxidative stress.
have been hospitalized or received intravenous drug therapy for
heart failure within the previous 12 months. The control group shows that oxygen/ozone protects the heart from acute myo-
will be treated with the best orthodox medications, while the cardial infarction and supports the need for performing a
experimental group will also receive ozone therapy, according clinical trial as described here.
to the classic and well-standardized procedure of major AHT.
Briefly, 200 mL of blood plus 22 mL of 3.8% sodium citrate will 7. Concluding Remarks
be exposed in an ozone-resistant glass bottle and gently mixed
for 5 minutes with 200 mL of a gas mixture composed of oxygen In this review, we aimed to draw attention to a rational bio-
and ozone precisely collected in a 50 mL syringe from a precise oxidative therapy, the biological reactions and effects of which
ozone generator with a direct photometric control. By consid- have been evaluated and proven to be valuable when tested in
ering the severity of the clinical conditions, it is proposed that PAD patients. We further aimed to stimulate the established
each patient receiving major AHT will undergo two treatments medical community to evaluate the potential value of ozone
every week for 4 months (i.e. a total of 32 treatments). The first therapy according to the 2008 Declaration of Helsinki. How-
two treatments will be performed with a low ozone concen- ever, it is regretful that several clinical trials to date that have
tration (20 mg/mL) to avoid excessive oxidative stress during the involved approximately 3000 patients have been performed
first week and will then be progressively increased up to using an irrational methodology. Millions of people suffer from
60 mg/mL for treatment after the first month. In other words, chronic limb, brain, and heart ischemia, which collectively
each patient should undergo the auto-reinfusion of a total represent the major cause of death worldwide. Such pathologies
of 6400 mL of ozonated blood over a period of 4 months. have a huge socioeconomic impact, particularly in the devel-
Although this is a minimal size trial, the number of AHT oping world. If the appropriate ozone therapy could be im-
treatments for 40 patients adds up to as many as 1280 auto- plemented in every public hospital, patients may have a better
transfusions, including 1280 sterile glass bottles of 500 mL each. prognosis and an improved quality of life.
The schedule of the ozone treatments is reported in table II.
Biochemical and instrumental tests and assessment of typi- Acknowledgments
cal end-points such as the comparative evaluation of morbidity, No funding was used in the preparation of this article. The authors have
mortality, ejection fraction and chronic inflammation para- no conflicts of interest that are relevant to the content of this article.
meters will be carried out before starting treatments, while final
results will be evaluated during the first 2 weeks of the fifth References
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